Methods for the detection of microbial contaminants

ABSTRACT

The invention provides methods and compositions for the detection and/or quantification of a microbial contaminant, for example, a bacterial endotoxin or a glucan, in a sample. In particular, the invention provides a test cartridge useful in the practice of hemocyte lysate-based assays for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides methods of making and using such cartridges. In addition, the invention provides a rapid, sensitive, multi-step kinetic hemocyte lysate-based assay for the detection and/or quantification of a microbial contaminant in a sample. In addition, the invention provides a glucan-specific lysate that can be used in a variety of assay formats, including, for example, a test cartridge, optionally configured to perform a kinetic assay.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/803,177, filed Mar. 17, 2004, which claims priority to andthe benefit of U.S. provisional patent application Ser. No. 60/455,632,filed Mar. 17, 2003, the contents of each of which are incorporated byreference herein.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions fordetecting and/or quantifying microbial contaminants in a sample. Moreparticularly, the invention relates to methods and compositions using ahemocyte lysate for detecting and/or quantifying microbial contaminationin a sample.

BACKGROUND OF THE INVENTION

Microbial contamination by, for example, Gram positive bacteria, Gramnegative bacteria, yeast, fungi, and molds may cause severe illness and,in some cases, even death in humans. Manufacturers in certainindustries, for example, the pharmaceutical, medical device, and foodindustries, must meet exacting standards to verify that their productsdo not contain levels of microbial contaminants that would otherwisecompromise the health of the recipient. These industries requirefrequent, accurate, and sensitive testing for the presence of suchmicrobial contaminants to meet certain standards, for example, standardsimposed by the United States Food and Drug Administration (USFDA) orEnvironmental Protection Agency. By way of example, the USFDA requirescertain manufacturers of pharmaceuticals and invasive medical devices toestablish that their products are free of detectable levels of Gramnegative bacterial endotoxin.

Furthermore, when people become infected with Gram negative bacteria,the bacteria may produce and secrete fever-inducing bacterialendotoxins. Bacterial endotoxins can be dangerous and even deadly tohumans. Symptoms of infection may range from fever, in mild cases, todeath. In order to promptly initiate proper medical treatment, itusually is important to identify as early as possible, the presence ofan endotoxin and, if possible, the concentration of the endotoxin in thepatient.

To date, a variety of assays have been developed to detect the presenceand/or amount of a microbial contaminant in a test sample. One family ofassays use hemocyte lysates prepared from the hemolymph of crustaceans,for example, horseshoe crabs. These assays typically exploit, in one wayor another, a clotting cascade that occurs when the hemocyte lysate isexposed to a microbial contaminant. A currently preferred hemocytelysate is amebocyte lysate (AL) produced from the hemolymph of ahorseshoe crab, for example, Limulus polyphemus, Tachypleus gigas,Tachypleus tridentatus, and Carcinoscorpius rotundicauda. Amebocytelysates produced from the hemolymph of Limulus, Tachypleus, andCarcinoscorpius species are referred to as Limulus amebocyte lysate(LAL), Tachypleus amebocyte lysate (TAL), and Carcinoscorpius amebocytelysate (CAL), respectively.

Routine assays that use LAL include, for example, gel clot assays, endpoint turbidometric assays, kinetic turbidometric assays, and endpointchromogenic assays (Prior (1990) “Clinical Applications of the LimulusAmebocyte Lysate Test” CRC PRESS 28-34). These assays, however, sufferfrom one or more disadvantages including reagent expense, assay speedand limited sensitivity ranges. Also, these assays typically requirethat samples be sent to a testing facility removed from the origin ofthe sample being tested. As a result, it may take hours to weeks beforea problem can be detected and remedied. Accordingly, there is an ongoingneed for faster and more sensitive methods, and portable test systemsemploying such methods, that overcome the need to submit samples to anoff-site testing facility.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery that it is possibleto make and use optical cartridges containing an immobilized hemocytelysate for use in hemocyte lysate-based assays. These cartridges may beused alone or in combination with optical detectors, for example, handheld optical detectors, to permit the assay of samples in the field,thereby obviating the need to send samples to an off-site testingfacility. Accordingly, the cartridges can be used in a point-of-use testsystem. In addition, the invention is based, in part, upon the discoveryof a rapid, sensitive, multi-step kinetic assay for determining thepresence and/or amount of microbial contaminant in a sample of interest.This type of assay can be implemented in a cartridge, or in any otherdesirable assay format. In addition, the invention is based, in part,upon the discovery of a glucan-specific hemocyte lysate and a method ofmaking such a lysate. The glucan-specific lysate may be incorporatedinto such a cartridge and/or used in a multi-step kinetic assay.

In one aspect, the invention provides a test cartridge for determiningthe presence and/or amount of a microbial contaminant in a sample. Thecartridge comprises a housing defining at least one fluid inlet port, atleast one optical cell, and at least one conduit having a fluidcontacting surface that connects and thus provides fluid flowcommunication between the fluid inlet port and the optical cell. Thecartridge further comprises a hemocyte lysate disposed on a first regionof the fluid contacting surface of the conduit, so that when a sample isapplied to the fluid inlet port, the sample traverses the region andsolubilizes the hemocyte lysate during transport of the sample-lysatemixture to the optical cell. This type of cartridge can be used toperform, for example, endpoint turbidometric and kinetic turbidometricassays.

The cartridge optionally may further comprise a chromogenic substratethat acts as a substrate for one or more of the enzymes in the hemocytelysate. The chromogenic substrate may be disposed in the first region,for example, combined with the hemocyte lysate. In this format, thesample resolubilizes or starts to resolubilize the hemocyte lysate andchromogenic substrate at substantially the same time. This type ofcartridge can be used to perform, for example, a kinetic chromogenicassay. Alternatively, the chromogenic substrate may be disposed on asecond region of the fluid contacting surface of the conduit, so thatwhen the sample moves along the conduit toward the optical cell itcontacts and reconstitutes the hemocyte lysate and chromogenic substrateat different regions of the conduit. In one embodiment, the secondregion is located downstream of the first region (i.e., the secondregion is located closer to the optical cell than the first region).This type of cartridge can be used to perform, for example, endpointchromogenic and multi-step kinetic chromogenic assays, as discussed inmore detail herein below.

In addition, a pre-selected amount of an agent representative of amicrobial contaminant, or “spike”, such as a bacterial endotoxin, a(1→3)-B-D glucan, or other microbial cell wall constituent, is disposedon a region of the fluid contacting surface of the conduit. Theinclusion of the agent or spike is particularly useful as it provides apositive control to demonstrate that an assay is working, and can alsodemonstrate whether an inhibitor or enhancer is present in the sample.The agent or spike may be disposed on the first region, the secondregion, or on another region of the conduit.

It is contemplated that the number of fluid inlet ports, optical cells,and conduits in a particular cartridge may vary depending on the numberof samples or microbial contaminants being tested at a particular time.A cartridge may be used to simultaneously assay duplicates of the samesample or simultaneously assay two or more different samples ofinterest. Alternatively, two or more different hemocyte lysates may bedisposed on the cartridge so that it is possible to determine thepresence and/or amount of two or more different microbial contaminantsat substantially the same time. In addition, several chromogenicsubstrates with different enzyme specificities and optical properties,for example, light absorption transmission, and/or fluorescentproperties, may be applied to the cartridge. This may permit thedetection of two or more different microbial contaminants atsubstantially the same time.

In one embodiment, the cartridge comprises a housing that defines (i) afirst fluid inlet port, a first optical cell, and a first conduit havinga fluid contacting surface that connects and thus provides fluid flowcommunication between the first fluid inlet port and the first opticalcell, and (ii) a second fluid inlet port, a second optical cell, and asecond conduit having a fluid contacting surface that connects and thusprovides fluid flow communication between the second fluid inlet portand the second optical cell. Within the housing, a first hemocyte lysateis disposed on a first region of the fluid contacting surface of thefirst conduit, so that when a sample is applied to the first fluid inletport, the sample traverses the region and reconstitutes and/orsolubilizes the first hemocyte lysate during transport to the firstoptical cell. Also within the housing, a second hemocyte lysate isdisposed on a first region of the fluid contacting surface of the secondconduit, so that when a sample is applied to the second fluid inletport, the sample traverses the region and reconstitutes and/orsolubilizes the second hemocyte lysate during transport to the secondoptical cell.

In one embodiment, a chromogenic substrate is disposed on a secondregion of the fluid contacting surface of the first conduit and/or achromogenic substrate is disposed on a second region of the fluidcontacting surface of the second conduit. In each embodiment, the secondregion preferably is located downstream of the first region in eachconduit. In another embodiment, different chromogenic substrates may bedisposed on fluid contacting surfaces of the first and second conduitsso that two different reactions may be monitored in the same cartridge.

In another embodiment, a pre-selected amount of an agent representativeof a microbial contaminant or spike is disposed on the fluid contactingsurface of the first or second conduit. The spike may be disposed on thefirst region or on another region of the conduit. The spike may beuseful as a positive control for the assay (i.e., indicates whether avalid test was run), and may also provide information on whether anenhancer or inhibitor is present in the sample.

As will be discussed in more detail below, the cartridges of theinvention may be adapted for use in a variety of different assays. Thepresence of a microbial contaminant may be indicative of, for example,past or present bacterial, yeast, fungal, or mold infection. It iscontemplated that, by using the appropriate hemocyte lysate, thecartridge may be used to detect the presence of a bacterial, yeast,fungal, or mold contaminant in a sample. The cartridges of the inventionare particularly useful at detecting the presence, and/or determiningthe amount, of a Gram negative bacterial endotoxin or glucan in asample.

During use, a sample to be tested is introduced into the sample inletport of the cartridge and is allowed to move to the optical cell.Movement of the sample along the conduit can be passive or can beinduced by an external force, for example, via positive or negativepressure in the conduit. For example, the sample can be pulled along theconduit to the optical cell via suction induced by a pump connected to apump port located downstream of the optical cell. A change in an opticalproperty of the sample is detected in the optical cell, the change beingindicative of the presence of a microbial contaminant in the sample. Inaddition, the time in which a pre-selected change occurs in an opticalproperty of the sample can be determined and compared against apredetermined standard curve to determine the concentration of themicrobial contaminant in the sample. The optical property may include acolor change, a change in absorbance or transmittance, a change inturbidity, a change in fluorescence or other change that can be detectedin a detector, spectrophotometer or the like. The change in the opticalproperty may be, for example, an increase in absorbance of light of apre-selected wavelength, or may be a decrease in transmission of lightof a pre-selected wavelength. As discussed below, the cartridge may beadapted to perform any one of a number of endpoint or kineticchromogenic, or turbidometric assays.

In another aspect, the invention provides methods of preparing thecartridge by disposing, for example, by drying, a hemocyte lysate onto asolid surface of at least one conduit of the cartridge. The hemocytelysate may then be reconstituted into an active form uponresolubilization of the hemocyte lysate. A volume of a mixturecomprising a hemocyte lysate of interest and a resolubilizing agentand/or an anti-flaking agent is applied to the surface of at least oneconduit and dried. The hemocyte lysate used will depend upon the assayfor which the cartridge will be used. The resolubilizing agent is anagent that, either alone or in combination with another resolubilizingagent, facilitates the resolubilization of one or more components of thehemocyte lysate once the lysate is exposed to a fluid sample. Theresolubilizing agent preferably also stabilizes the lysate in its driedform. The resolubilizing agent provided in the mixture facilitates thestability of the reagents and their dissolution during the assay.Resolubilizing agents include, for example, one or more sugars, salts,or combinations thereof. Preferred sugar resolubilizing agents include,for example, mannitol, mannose, sorbitol, trehalose, maltose, dextrose,sucrose, and other monosaccharides or disaccharides. The anti-flakingagent included in the mixture further stabilizes the reagents andreduces flaking of the dried lysate. The anti-flaking agent preferablyalso stabilizes the lysate in its dried form. Preferred anti-flakingagents include, for example, one or more polymers, for example,polyethylene glycol, polyvinyl pyrolidone, polyvinyl alcohol, mannitol,dextran, and proteins, for example, serum albumin.

In one embodiment, the lysate/resolubilizing agent/anti-flaking agentmixture is disposed in a first region of at least one conduit of thecartridge. The mixture then is dried onto a surface of the conduit in anenvironment having a temperature of about 4° C. to about 40° C., morepreferably, from about 10° C. to about 35° C., more preferably, fromabout 15° C. to about 30° C. and a relative humidity of about 0% toabout 30%, more preferably, from about 2% to about 20%, more preferably,from about 4% to about 10%. Preferably, the temperature is about 25° C.and the relative humidity is about 5%. Drying preferably occurs forabout 10 minutes to about 8 hours, more preferably for about 1 hour in atemperature regulated drying chamber. The lysate/resolubilizingagent/anti-flaking agent mixture optionally may also include an agentrepresentative of a microbial contamination (i.e., a spike), forexample, a bacterial endotoxin, a (1→3)-B-D glucan or other microbialcell wall constituents.

In another embodiment, the mixture is dried onto the surface of theconduit by lyophilization or freeze drying, for example, at temperaturesbelow 0° C., for example, from about −75° C. to about −10° C., morepreferably from about −30° C. to about −20° C.

In addition, a chromogenic substrate, comprising a resolubilizing agentand an anti-flaking agent is applied to a second region of the cartridgeand dried onto the cartridge as described above. The chromogenicsubstrate may comprise an anti-frothing agent, for example, polyvinylalcohol and polypropylene glycol.

Although the drying procedure is discussed in relation to a testcartridge, the drying procedure can be used to dry the lysate onto avariety of different solid supports. For example, the method can also beused to dry the lysate on the surface of a well disposed within ordefined by a solid support, for example, a 12-well or a 96-well plate.

In another aspect, the invention provides an improved method referred toas a two-step or multi-step kinetic assay for detecting the presenceand/or quantifying the amount of a particular microbial contaminant in asample. The sample to be tested is contacted with a hemocyte lysatecomprising an activatable enzyme, for example, a pro-clotting enzyme ora clotting enzyme, that is activated if the microbial contaminant ispresent in the sample. After contacting the sample with the lysate, thesample-lysate mixture is incubated for a preselected period of time.Then, the sample-lysate mixture is contacted with a substrate, forexample, a chromogenic substrate, for the activated enzyme. If thesample-lysate substrate mixture contains an activated enzyme, theactivated enzyme produces a change in the substrate, which in turnproduces a predetermined change in an optical property of thesample-lysate-substrate mixture. The time in which the predeterminedchange occurs then is determined. The resulting time then is compared toa predetermined standard curve to determine whether the microbialcontaminant is present in the sample and/or to determine the amount ofmicrobial contaminant present in the sample. For example, theconcentration of microbial contaminant in a sample can be measured bycomparing the time required to produce the predetermined change in theoptical property against a predetermined standard curve of the microbialcontaminant. Using this type of assay, it is possible to adjust thetiming of the steps to produce an assay of predetermined sensitivity andduration.

The optical property measured can be a change (e.g., an increase ordecrease) in an optical property, for example, absorbance at aparticular wavelength, transmittance at a particular wavelength,fluorescence at a particular wavelength, or optical density. Forexample, the optical property may be a change in absorbance ortransmittance at a wavelength in the range from about 200 nm to about700 nm, and more preferably in the range from about 350 nm to about 450nm.

The chromogenic substrate may be any substrate for a lysate enzyme thatis activated (e.g., hydrolyzed) to cause a detectable chromogenic orfluorogenic change, for example, by release of a chromophore or afluorophore, that is detectable by an optical detector. In oneembodiment, the chromogenic substrate for LAL contains apara-nitroaniline chromophore, such as that, for example, in thechromogenic substrate acetate-Ile-Glu-Ala-Arg-pNA. The proteases in theLAL cleave colorless tetrapeptide to release the pNA group, which causesa color change. Cleavage of the tetrapeptide simulates the cleavagereaction of the proteases in the LAL with coagulogen, a clottingcomponent that contains the tetrapeptide. As a result, by using thischromophore, it is possible to measure the progress of the reaction bymeasuring the change in optical density at about 395 nm. Otherchromophores may include dinitrophenyl alanine, cyclohexyl alanine andthe like. Alternatively, the substrate may contain a fluorophore, forexample, 7-amino-4-methyl coumarin, 7-amino-4-trifluoromethyl coumarin,and 4-methoxy-2-naphthalyamine. Fluorogenic substrates for LAL thatcontain N-methylcoumarin as a leaving group are available from EnzymeSystems Products, Livermore, Calif.

The multi-step kinetic assay may be performed in a variety of formats,for example, in a tube, cuvette, cartridge, well on a solid support(such as a 96-well multi-well plate), or other vessel suitable for usein combination with an optical detector, for example, aspectrophotometer, fluorimeter, luminometer, or the like.

In another aspect, the invention provides a method for producing anamebocyte lysate depleted of Factor C activity. The method comprises thesteps of: (a) providing a preparation of amebocytes; and (b) lysing theamebocytes in the presence of at least 0.05M salt to provide anamebocyte lysate preparation depleted of Factor C activity. The methodoptionally includes the step of, after step (b), removing cellulardebris, for example, cell membranes, and then harvesting the remaininglysate. The cellular debris may be sedimented by centrifugation and theremaining supernatant harvested.

In one embodiment, the salt may comprise a monovalent cation, forexample, a sodium or potassium salt. Salts useful in the practice of theinvention include, for example, sodium chloride, potassium chloride,sodium acetate, and potassium acetate. The salt concentration can be inthe range from 0.15 M to about 6 M, more preferably from about 0.25 M toabout 4 M, and more preferably from about 1 M to 2 M. However, theprecise concentration of salt necessary to remove or reduce Factor Cactivity may be determined by routine experimentation. For example,amebocytes can be lysed in the presence of difference concentrations ofsalt, and the residual lysates can then be checked to see whether acoagulin clot forms in the presence of a bacterial endotoxin. Theforegoing method can produce a glucan-specific amebocyte lysate that issubstantially free of Factor C activity. The lysate, therefore, retainsFactor G activity but is depleted of Factor C activity.

In another aspect, the invention provides an amebocyte lysatesubstantially free of Factor C activity, wherein the lysate comprises atleast about 0.25M salt and wherein the lysate is capable of producing acoagulin gel in the presence of glucan. The lysate may comprise, fromabout 0.25 M salt to about 6 M salt, from about 0.5 M salt to 4 M salt,and from about 1 M to about 2 M salt. In one embodiment, the saltcontains a monovalent cation, for example, a sodium ion or a potassiumion. For example, the salt may include sodium chloride, potassiumchloride, sodium acetate, potassium acetate, or a combination thereof.

The foregoing and other objects, features and advantages of the presentinvention will be made more apparent from the following drawings anddetailed description of preferred embodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be better understood byreference to the drawings described below in which,

FIG. 1 is a schematic representation of the coagulation system presentin amebocytes;

FIGS. 2A-2D are schematic illustrations of an exemplary cartridge of theinvention in perspective view (FIG. 2A), top view (FIG. 2B), side view(FIG. 2C), and end view (FIG. 2D);

FIGS. 3A-3B are schematic illustrations of an exemplary cartridge of theinvention wherein each conduit has a separate fluid inlet port (FIG.3A), and wherein two conduits share a single common fluid inlet port(FIG. 3B);

FIGS. 4A-4B are schematic illustrations of an exemplary cartridge of theinvention wherein each conduit has its own fluid inlet port (FIG. 4A),and wherein a pair of conduits share a single common fluid inlet port(FIG. 4B);

FIGS. 5A-5D are schematic illustrations of an exemplary cartridge inwhich FIG. 5A is a view of a bottom half of an exemplary cartridge ofthe invention showing the locations of immobilized hemocyte lysate andchromogenic substrate, FIG. 5B is a view of a top half of an exemplarycartridge of the invention showing the location of an immobilized agentrepresentative of a microbial contaminant (i.e., spike), FIG. 5C is across-sectional view of the fabricated cartridge through section A-A,and FIG. 5D is a cross-sectional view of the fabricated cartridgethrough section B-B;

FIG. 6A-6D are schematic illustrations of two exemplary cartridges, inwhich FIG. 6A is a top view of a first embodiment of a cartridge, FIG.6B is a side view of the first embodiment of the cartridge pictured inFIG. 6A, FIG. 6C is a top view of a second, different embodiment of acartridge, and FIG. 6D is a side view of the second embodiment of thecartridge of FIG. 6C;

FIG. 7 is a flow chart for an exemplary multi-step kinetic chromogenicassay of the invention;

FIG. 8A-8B are schematic illustrations showing a cartridge incombination with an exemplary optical detector in which FIG. 8A showsthe cartridge being inserted into the detector, and FIG. 8B shows thecartridge actually inserted into the detector;

FIG. 9 is a graphical representation showing changes in light absorbanceor optical density (full line) or light transmittance (dashed line)through an optical cell of an exemplary cartridge of the inventionduring a multi-step kinetic chromogenic assay;

FIGS. 10A-10B are graphical representations of absorbance values in anend point chromogenic assay where FIG. 10A shows the absorbance valuesof endotoxin standards (1.0 Endotoxin Units (EU)/mL (A1), 0.5 EU/mL(A2), 0.25 EU/mL (A3), and 0.125 EU/mL (A4)) over time in an endpointchromogenic assay performed in a cartridge of the invention, and FIG.10B shows a standard curve generated by plotting the absorbance valuesof each concentration of endotoxin at T=780 seconds;

FIGS. 11A-11D are graphical representations of absorbance values for akinetic chromogenic assay performed in a cartridge of the invention,where FIG. 11A shows the absorbance values of a 5.0 EU/mL endotoxinstandard, FIG. 11B shows the absorbance values of a 0.5 EU/mL endotoxinstandard, FIG. 11C shows the absorbance values of a 0.05 EU/mL endotoxinstandard, and FIG. 11D is a standard curve generated by plotting the logof endotoxin concentration (X-axis) versus the log of absorbance valueat onset time (Y axis);

FIG. 12 is a graphical representation of a standard curve for amulti-step kinetic chromogenic assay performed in a cartridge of theinvention, generated by plotting the log of endotoxin concentration(X-axis) versus the log of absorbance value at onset time (Y axis);

FIG. 13 is a graphical representation of a standard curve for asingle-step kinetic chromogenic assay performed in a microtiter plate,generated by plotting the log of endotoxin concentration (X-axis) versusthe log of absorbance value at onset time (Y axis);

FIG. 14 is a graphical representation of a standard curve for anendpoint chromogenic assay performed in a microtiter plate, generated byplotting the absorbance values (Y axis) of each concentration ofendotoxin (X axis); and

FIG. 15 is a graphical representation of a standard curve for amulti-step kinetic chromogenic assay performed in a microtiter plate,generated by plotting the log of endotoxin concentration (X-axis) versusthe log of absorbance value at onset time (Y axis).

FIGS. 16A-B are graphical representations of amebocyte lysate kineticreactions for standard LAL and glucan-specific LAL. FIG. 16A representsassays performed using lipopolysaccharide, and FIG. 16B representsassays performed using glucan. Row A in each Figure represents standardLAL containing both the Factor C and Faction G cascades, Row B in eachFigure represents glucan-specific lysate (i.e., Factor G-specificlysate) prepared by lysing amebocytes in 1M sodium chloride and Row Crepresents glucan-specific lysate prepared by lysing amebocytes in 2MNaCl. Columns 1-5 represent several dilutions of the lipopolysaccharide(10 ng/ml, ng/ml, 100 pg/ml, 10 pg/ml, 0) or glucan (100 μg/ml, 10μg/ml, 1 μg/ml, 100 ng/ml, 0) added to each sample, wherein theconcentration decreases from column 1 to column 5;

FIG. 17 is a graphical representation of a logarithmic plot of a glucanstandard curve obtained using a multi-step kinetic assay;

FIG. 18 is a graphical representation of an assay measuring thefluorescence emitted from different concentrations of a fluorogenicsubstrate Glu-Gly-Arg-AMC in a multi-step kinetic assay in the cartridgeformat;

FIG. 19 is a graphical representation showing the proportionaldisplacement of various concentrations of lipopolysaccharide-fluoresceinisothiocyanate (1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 100 pg/ml, 10pg/ml, and control) by different dilutions of a fluorescein-labeledligand (1/50, 1/150, and 1/450).

In the drawings, which are not necessarily drawn to scale, likecharacters refer to the same or similar parts throughout the Figures.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an optical cartridge containing an immobilizedhemocyte lysate for use in hemocyte-lysate based assays. Thesecartridges may be used alone or together with an optical detector, forexample, a hand held optical detector. In addition, the inventionprovides a rapid, sensitive, broad range, multi-step assay that isuseful in determining the presence and/or amount of a microbialcontaminant in a sample. Although the cartridge and method may be usedseparately, they are particularly effective when combined together toprovide a system that can be used in the field to provide rapid testresults. This facilitates quicker elimination and/or treatment ofmicrobial contamination. In addition, the invention provides a Factor Cspecific lysate for detecting the presence and/or amount of glucan in asample. The lysate, therefore, can be used to determine the presenceand/or amount of a yeast or mold contaminant in a sample.

The Cartridge

It is contemplated that the cartridges of the invention may beformulated with one or more hemocyte lysates and used in a variety ofassays to detect the presence and/or amount of a microbial contaminantin a sample. A number of hemocyte lysate-based assays for the detectionand/or quantification of a microbial contaminant can be performed in thecartridge of the invention, for example, as illustrated in FIG. 2. Thecartridge may be used on its own and the test result detected by eye ormay be used in combination with an optical detector, for example, ahand-held optical detector as shown and described in U.S. Pat. No. Des.390,661.

By way of example and as illustrated in FIGS. 2A-2D, cartridge 1 has asubstantially planar housing fabricated, for example, from a moldablebiocompatible material. The housing may be fabricated from any material,however, transparent and/or translucent glass or polymers are preferred.Preferred polymers include, for example, polystyrene, polycarbonate,acrylic, polyester, optical grade polymers, or any plastic such that theoptical cell is substantially transparent. The housing contains at leastone fluid inlet port 4, at least one optical cell 6, and at least oneconduit 8 having a fluid contacting surface for providing fluid flowcommunication between the fluid inlet port 4 and optical cell 6. Theonly requirements for the optical cell 6 are that it defines a voidcapable of containing a sample to be tested and that a portion of theoptical cell 6 is transparent to light. Cartridge 1 may also have atleast one pump port 12 in fluid flow communication with fluid inlet port4 and optical cell 6 for attaching the cartridge 1 to a pump. The pumpmay then impart a negative pressure via pump port 12 to pull the samplefrom fluid inlet port 4 to optical cell 6. A hemocyte lysate is disposedon a first region 14 of the fluid contacting surface of conduit 8, sothat when a sample is applied to fluid inlet port 4, the sampletraverses region 14 and solubilizes or reconstitutes the hemocyte lysateinto the sample as it moves toward optical cell 6. This type ofcartridge 1 may be used for performing, for example, an endpointturbidometric or a kinetic turbidometric assay. In an embodiment, achromogenic substrate may also optionally be applied to the surface ofthe conduit 8 at first region 14 together with the hemocyte lysate. Thistype of cartridge 1 may be used for performing, for example, a kineticchromogenic assay.

In a preferred embodiment, as illustrated in FIGS. 2A-2D, a secondregion 16 of the fluid contacting surface of conduit 8 is spaced apartfrom and downstream of first region 14. In this configuration, hemocytelysate is disposed at first region 14 and a chromogenic substrate isdisposed at second region 16, so that after the sample is contacted withthe hemocyte lysate in region 14, the sample-lysate mixture traversesconduit 8 and contacts the chromogenic substrate in region 16. Thesample-lysate-substrate mixture then traverses conduit 8 to optical cell6. This type of cartridge may be used for performing, for example, anendpoint chromogenic assay or a multi-step kinetic chromogenic assay, asdiscussed in more detail below.

Depending upon the type of assay to be performed, a pre-selected amountof an agent representative of a microbial contaminant, or “spike,” suchas a bacterial endotoxin, is disposed on first region 14 of the fluidcontacting surface of one or more conduits 8. Alternatively, the spikemay be disposed on a different region of the conduit 8.

It is contemplated that the cartridge 1 may have a variety of differentconfigurations, demonstrated, for example, in FIGS. 3 and 4. FIG. 3Ashows a cartridge 1 comprising two conduits 8 with each conduit 8 havingits own sample inlet port 4. FIG. 3B shows a cartridge 1 comprising twoconduits 8 with each conduit 8 sharing a common sample inlet port 4.FIG. 4A shows a cartridge 1 comprising four separate conduits 8 witheach conduit 8 having its own sample inlet port 4. FIG. 4B shows acartridge 1 comprising two pairs of conduits 8, with each pair havingits own sample inlet port 4.

The cartridges can be designed and used according to the type and/ornumber of tests required. For example, a single sample may be tested,for example, in duplicate or triplicate, for example, for researchlaboratory uses or for medical device and biopharmaceutical testing.Alternatively, two or more different samples may be tested individually,for example, for dialysis facility testing of water and dialysate. Thecartridge preferably is a single-use, disposable cartridge that isdiscarded after one use. The cartridge of the invention can useapproximately 20-100 fold less hemocyte lysate per sample than is usedin the conventional endpoint chromogenic or kinetic chromogenic assaysperformed in multi-well plates, and thus provides a less costly andenvironmentally-friendlier test.

Once a particular assay format has been chosen, the cartridge may befabricated as discussed below.

Cartridge Fabrication

All the reagents and materials used to prepare the cartridge preferablyare free of the microbial contaminant for which the cartridge ultimatelywill be used to test.

It is contemplated that the cartridge may be fabricated with anyhemocyte lysate of interest. As used herein, the term, “hemocyte lysate”is understood to mean any lysate or a fraction or component thereof,produced by the lysis and/or membrane permeabilization of hemocytes, forexample, amebocytes and hemolymph cells, (i) extracted from a crustaceanor insect and/or (ii) cultured in vitro after extraction from the host.Hemocyte cellular material that has been extruded from hemolymph cellsby contact with a membrane permeabilization agent such as a Ca²⁺ionophore or the like (i.e., extruded other than by lysis) or otherwiseextracted without cellular lysis is also considered to be a hemocytelysate. A preferred hemocyte lysate is an amebocyte lysate prepared fromthe blood of a crustacean, for example, a horseshoe crab or Jonah crab.It is also contemplated that hemocyte lysate may include a cocktail ofone or more natural (e.g., purified) or synthetic components of theenzyme cascades shown in FIG. 1.

As used herein, the term “amebocyte lysate” is understood to mean anylysate or fraction or component thereof produced by the lysis,extrusion, or extraction of the cellular contents from amebocytesextracted from a crustacean, for example, a horseshoe crab. Theamebocyte lysate comprises at least one component of an enzymaticcascade (for example, as shown in FIG. 1) and/or produces a clot in thepresence of an endotoxin, for example, a Gram negative bacterialendotoxin and/or a glucan, for example, a (1→3)-β-D glucan, produced bya yeast or a mold. Preferred amebocyte lysates can be derived fromhorseshoe crabs, which include crabs belonging to the Limulus genus, forexample, Limulus polyphemus, the Tachypleus genus, for example,Tachypleus gigas, and Tachypleus tridentatus, and the Carcinoscorpiusgenus, for example, Carcinoscorpius rotundicauda.

Crude lysates may be produced using the procedure as originallydescribed in Levin et al. (1968) THROMB. DIATH. HAEMORRH. 19: 186, withmodification, or in Prior (1990) “Clinical Applications of the LimulusAmebocyte Lysate Test” CRC PRESS 28-36 and 159-166, and in U.S. Pat. No.4,322,217. Other lysates may include those, for example, described inU.S. Pat. Nos. 6,270,982 and 6,391,570.

Presently, LAL is employed as the amebocyte lysate of choice in manybacterial endotoxin assays because of its sensitivity, specificity, andrelative ease for avoiding interference by other components that may bepresent in a sample. LAL, when combined with a sample containingbacterial endotoxin and optionally with certain LAL substrates, reactswith the endotoxin in the sample to produce a detectable product, suchas a gel, increase in turbidity, or a colored or light-emitting product,in the case of a synthetic chromogenic substrate. The product may bedetected, for example, either visually or by the use of an opticaldetector.

As shown in FIG. 1, the coagulation system of hemolymph, like themammalian blood coagulation system, comprises at least two coagulationcascades that include an endotoxin-mediated pathway (the Factor Cpathway) and a (1→3)-B-D glucan-mediated pathway (the Factor G pathway).See, for example, Morita et al. (1981) FEBS LETT. 129: 318-321 andIwanaga et al. (1986) J. PROTEIN CHEM. 5: 255-268.

The endotoxin-mediated activation of LAL is well understood and has beenthoroughly documented in the art. See, for example, Levin et al. (1968)supra; Nakamura et al. (1986) EUR. J. BIOCHEM. 154: 511; Muta et al.(1987) J. BIOCHEM. 101: 1321; and Ho et al. (1993) BIOCHEM. & MOL. BIOL.INT. 29: 687. When bacterial endotoxin is contacted with LAL, theendotoxin initiates a series of enzymatic reactions, referred to in theart as the Factor C pathway, that can involve three serine proteasezymogens called Factor C, Factor B, and pro-clotting enzyme (see, FIG.1). Briefly, upon exposure to endotoxin, the endotoxin-sensitive factor,Factor C, is activated. Activated Factor C thereafter hydrolyses andactivates Factor B, whereupon activated Factor B activates proclottingenzyme to produce clotting enzyme. The clotting enzyme thereafterhydrolyzes specific sites, for example, Arg¹⁸-Thr¹⁹ and Arg⁴⁶-Gly⁴⁷ ofcoagulogen, an invertebrate, fibrinogen-like clottable protein, toproduce a coagulin gel. See, for example, U.S. Pat. No. 5,605,806.

It is possible to produce an endotoxin-specific lysate by removingFactor G activity from the lysate, such as the Factor G depleted lysatesproduced by the methods described in U.S. Pat. Nos. 6,391,570 and6,270,982. Also, it is contemplated that lysates may be depleted ofFactor G activity by the addition of certain inhibitors or modulators ofFactor G activity, for example, certain detergents, saccharides,polysaccharides, and other reagents described in U.S. Pat. Nos.5,155,032; 5,179,006; 5,318,893; 5,474,984; 5,641,643; 6,270,982; and6,341,570. An endotoxin-specific lysate is a lysate capable of reactingwith a bacterial endotoxin but in which the reactivity to (1→3)-B-Dglucan has been depleted by at least 80%, more preferably by at least90%, and more preferably by at least 95% relative to the crude lysatefrom which the endotoxin-specific lysate was prepared.

(1→3)-B-D glucans and other LAL reactive glucans, produced bymicroorganisms such as yeasts and molds, can also activate the clottingcascade of LAL, through a different enzymatic pathway, referred to inthe art as the Factor G pathway (see, FIG. 1). Factor G is a serineprotease zymogen that becomes activated by (1→3)-β-D glucan or other LALreactive glucans. Upon exposure to (1→3)-β-D glucan, for example, FactorG is activated to produce activated Factor G. Activated Factor Gthereafter converts the proclotting enzyme into clotting enzyme,whereupon the clotting enzyme converts coagulogen into coagulin.

As used herein, the term, “(1→3)-β-D glucan” is understood to mean anywater soluble polysaccharide, disaccharide or derivative thereof that is(i) capable of inducing formation of a coagulin clot in crude Limulusamebocyte lysate, and (ii) contains at least two β-D glucosides,connected by a (1→3)-β-D glycosidic linkage (see Formula I). It iscontemplated that such a polysaccharide or derivative thereof, inaddition to containing a (1→3)-β-D glycosidic linkage, may also containglucoside moieties connected by a variety of other glycosidic linkages,for example, via a (1→4)-β-D glycosidic linkage and/or by a (1→6)-β-Dglycosidic linkage. It is contemplated that such (1→3)-β-D glucans maybe isolated from a variety of sources including, without limitation,plants, bacteria, yeast, algae, and fungi, or alternatively may besynthesized using conventional sugar chemistries.

It is possible to produce a (1→3)-B-D glucan specific lysate byproducing a lysate depleted of Factor C activity. As shown herein, it ispossible to produce a glucan-specific lysate by lysing amebocytes in thepresence of at least 0.15 M salt, more preferably 0.25 M salt, forexample, a salt containing a monovalent cation, such as sodium orpotassium ions. For example, the amebocytes are lysed in about 0.15 M toabout 6 M salt, for example, sodium chloride. Alternatively, theamebocytes are lysed in a solution containing from about 0.25 M to about4 M salt, or from about 1 M to about 2M salt. When using sodiumchloride, it appears that the amoeboctye preparation loses substantialFactor C activity when the amebocytes are lysed in a solution containing0.25 M sodium chloride. However, the concentration of other saltsnecessary to produce a comparable results may be determined by routinetitration experiments. For example, amebocytes may be lysed in differentconcentrations of salt and the resulting lysates examined for theirability to produce a coagulin gel in the presence of a Gram negativebacterial endotoxin. The concentration of salt may be chosen where theresulting lysate has lost a substantial amount of reactivity to thebacterial endotoxin. Other salts that may be used include, but are notlimited to, monovalent ionic salts, such as, potassium chloride,potassium acetate and sodium acetate. An exemplary method for producinga glucan specific lysate is described in Example 4. A glucan-specificlysate is a lysate capable of reacting with glycan, for example,(1→3)-β-D glucan, but in which reactivity to a bacterial endotoxin orlipopolysaccharide has been depleted by at least 80%, more preferably atleast 90%, and more preferably at least 95% relative to the crude lysatefrom which the glucan-specific lysate was prepared.

Methods for enhancing the sensitivity of hemocyte lysate for endotoxin,for example, may include, without limitation, aging the crude hemocytelysate, adjusting pH, adjusting the concentration of divalent cations,adjusting the concentration of coagulogen, chloroform extraction, andthe addition of serum albumin, biocompatible buffers and/or biologicaldetergents.

As will be apparent to one of ordinary skill, divalent metal salts,which are known to promote activation of the pro-clotting enzyme ofhemocyte lysate, as well as buffers to avoid extremes of pH that couldinactivate the clotting enzyme preferably are included in the lysate.Any of the buffers and salts that are understood in the art to becompatible with the amebocyte lysate system may be used. Typicalformulation additives may include, without limitation, about 100-300 mMNaCl, about 10-100 mM divalent cations (e.g., Mg²⁺ or Ca²⁺),biocompatible buffers, e.g., Tris (tris(hydroxy)aminomethane), to give afinal pH of about 6.0 to about 8.0, and, if the lysate is to be freezedried, then sugars, e.g., mannitol or dextran. It is contemplated thatthe choice of appropriate formulation additives may also be determinedby routine experimentation.

Synthetic chromogenic substrates have been used to measure the level ofendotoxin-activated pro-clotting enzyme in LAL prepared from thehemolymph of both Tachypleus tridentatus and Limulus polyphemushorseshoe crabs (Iwanaga et al. (1978) HEMOSTASIS 7: 183-188). During anLAL assay that uses a chromogenic substrate, the pro-clotting enzyme (aserine protease) in the LAL is activated by endotoxin and cleaves thesubstrate's peptide chain on the carboxyl side of arginine so as torelease the chromogenic group from the substrate, thereby releasing amarker compound that can be easily detected by, for example,spectrophotometry. One advantage of using a synthetic chromogenicsubstrate in an LAL assay in place of a conventional LAL gelation testis that the amount of activated clotting enzyme can be quantified andcorrelated to endotoxin levels in the sample.

Any chromogenic substrate that is cleaved by the clotting enzyme of ahemocyte lysate may be used in the practice of the invention. U.S. Pat.No. 5,310,657, for example, describes an exemplary chromogenic substratehaving the formula R₁-A₁-A₂-A₃-A₄-B-R₂, where R₁ represents hydrogen, ablocking aromatic hydrocarbon or an acyl group; A₁ represents an L orD-amino acid selected from Ile, Val or Leu; A₂ represents Glu or Asp; A₃represents Ala or Cys; A₄ represents Arg; B represents a linkageselected from an ester and an amide; and R₂ represents a chromogenic offluorogenic group which is covalently attached to the C-carboxylterminal of Arginine through the B linkage, the fluorogenic orchromogenic moiety being capable of being cleaved from the remainder ofthe chromogenic substrate to produce a chromogen or a fluorogen. Anexemplary chromogenic substrate has the consensus sequenceacetate-Ile-Glu-Ala-Arg-pNA, where pNA represents a para-nitroanilinegroup. U.S. Pat. No. 4,188,264 describes a peptide substrate with astructure consisting of L-amino acids in the sequence R₁-Gly-Arg-R₂where R₁ represents an N-blocked amino acid and R₂ is a group that canbe released by enzymatic hydrolysis to yield a colored compound, HR₂.U.S. Pat. No. 4,510,241 discloses a chromogenic peptide substrate, whichdiffers from the previous substrate in that the Gly moiety is replacedin the sequence by Ala or Cys. Alternatively, the chromogenic substratemay contain a fluorophore, for example, 7-amino-4-methyl coumarin,7-amino-4-trifluoromethyl coumarin, and 4-methoxy-2-naphthalyamine.

Inhibition or enhancement of the assay occurs when substances in thetest sample interfere with the hemocyte lysate reaction. Inhibitionresults in a longer reaction time, indicating lower levels of microbialcontamination than may actually be present in the test sample.Enhancement results in shorter reaction time, indicating higher levelsof microbial contamination than may actually be present in the testsample. To verify the lack of inhibition or enhancement, an aliquot oftest sample (or a dilution of the test sample) is “spiked” with a knownamount of an agent representative of the microbial contaminant to bemeasured. It is recommended that the microbial contaminant spike resultsin a final microbial contaminant concentration in the sample equal tothe mid-point, on a log basis, between the microbial contaminantconcentration of the highest and lowest standards in the standard curve.For example, in an assay with a standard curve spanning from 50Endotoxin Units (EU)/mL to 0.005 EU/mL, samples should be spiked tocontain a final microbial contaminant concentration of 0.5 EU/mL. In anassay with a standard curve spanning from 1 EU/mL to 0.01 EU/mL, themicrobial contaminant spike should result in a final microbialcontaminant concentration of 0.1 EU/mL.

The spiked sample is assayed in parallel with the unspiked sample. Theresulting microbial contaminant concentration in the unspiked sample andthe microbial contaminant recovered in the spiked sample then arecalculated. The microbial contaminant recovered should equal the knownconcentration of the spike within about 25%. If the test sample (ordilution) is found to inhibit or enhance the reaction, the sample mayrequire further dilution until the inhibition or enhancement isovercome. Initially, one may want to screen for inhibition orenhancement by testing 10-fold dilutions of test sample. Once theapproximate non-inhibitory or non-enhancing dilution is determined, theexact dilution can be found by testing two-fold dilutions around thisdilution. The degree of inhibition or enhancement will be dependent uponthe concentration of the test sample. If several concentrations of thesame sample are to be assayed, it is necessary to establish performancecharacteristics for each concentration independently.

In fabricating the cartridge of the invention, it is helpful to combinethe amebocyte lysate and chromogenic substrate with at least oneresolubilizing agent, such as a sugar or salt, and at least oneanti-flaking agent, such as a polymer, prior to drying the lysate ontothe solid support.

The resolubilizing agent preferably stabilizes the lysate in the driedform and facilitates resolubilization of the reagents during the assay.Useful resolubilizing agents include, for example, mannitol, mannose,sorbitol, trehalose, maltose, dextrose, sucrose, and othermonosaccharides and disaccharides. The hemocyte lysate and chromogenicsubstrate preferably contain from about 0.01% (w/v) to about 20% (w/v),more preferably from about 0.1% (w/v) to about 1.0% (w/v) of theresolubilizing agent prior to drying.

The anti-flaking agent is an agent that prevents or reduces thelikelihood that the hemocyte lysate and/or chromogenic substrate becomesdisassociated from a solid support in the form of a dry flake. Theanti-flaking agent preferably also stabilizes the hemocyte lysate orchromogenic substrate in the dried form. Useful anti-flaking agentsinclude, for example, one or more polymers, including, for example,polyethylene glycol, polyvinyl pyrolidone, dextrans, mannitol, andproteins, for example, serum albumin. The lysate preferably containsfrom about 0.01% (w/v) to about 25% (w/v), more preferably from about0.1% (w/v) to about 1.0% (w/v) of anti-flaking agent prior to drying.

In addition, it has been found that certain polymers reduce theformation of air bubbles (e.g., frothing) when the hemocyte lysateand/or chromogenic substrate are resolubilized. Useful anti-frothingagents include polyvinyl alcohol and polypropylene glycol. In order toreduce frothing, the lysate and/or chromogenic substrate may containfrom about 0.01% (w/v) to about 10% (w/v), more preferably from about0.1% (w/v) to about 1.0% (w/v) anti-frothing agent prior to drying.

An exemplary fabrication process for the cartridge is described withreference to FIG. 5, in which FIG. 5A represents a bottom half 2 ofcartridge 1 and FIG. 5B represents a top half 3 of cartridge 1. Onceprepared, the two halves of the cartridge 1 are joined to one another byadhesive, solvent bonding, ultrasonic welding, snap fit joints, or thelike.

In FIG. 5A, the bottom half 2 of the cartridge 1 defines one half ofeach conduit 81 (each having a first region 14′ and a second region16′). During fabrication of the bottom half 2 of the cartridge 1,hemocyte lysate is applied to each first region 14′ and chromogenicsubstrate is applied to each second region 16′. In FIG. 5B, the top half3 of the cartridge 1 defines one half of each conduit 8″. Duringfabrication of top half 3 of the cartridge 1, an agent representative ofa microbial contaminant (i.e., a spike), for example, a preselectedamount of an endotoxin, is applied to region 14″. Once the reagents havebeen applied to the respective top 3 and bottom 2 halves of thecartridge 1, the cartridge halves 2 and 3 then are dried underconditions that preserve the activity of the hemocyte lysate and permitreconstitution of the hemocyte lysate to produce active lysate. In orderto preserve the activity of the reagents during drying, the cartridgehalves 2 and 3 are placed in an environment having a temperature fromabout 4° C. to about 40° C., more preferably, from about 10° C. to about35° C., more preferably, from about 15° C. to about 30° C., and arelative humidity from about 0% to about 30%, more preferably, fromabout 2% to about 20%, more preferably from about 4% to about 10%.Preferred drying conditions include a temperature of about 25° C. and arelative humidity of about 5%. An exemplary protocol for manufacturing acartridge of the invention is provided in Example 1.

In an alternative approach, the hemocyte lysate may be dried via freezedrying under standard conditions, about −30° C. to about −40° C. undervacuum.

After drying, the two cartridge halves 2 and 3 are joined to one anotherto create an intact cartridge 1. FIG. 5C is a cross-sectional viewthrough Section A-A′ in which the two halves of the conduit (namely 8′and 8″) together create an intact conduit 8, wherein region 14′ of thebottom 8′ of each conduit contains immobilized hemocyte lysate 20 andregion 14″ of the top 8″ of one conduit contains immobilized endotoxin22.

FIG. 5D is a cross-sectional view through Section B-B′ in which region16′ of the bottom 8′ of each conduit contains immobilized chromogenicsubstrate 24.

FIGS. 6A-6D are illustrations of two exemplary cartridges 1 of theinvention corresponding to FIGS. 6A-6B and FIGS. 6C-6D. In FIG. 6A, thecartridge 1 may have an alternative finger grip 5 as shown with thedashed line. FIGS. 6A and 6B illustrate that the optical cell 6 in thefirst cartridge 1 is substantially cylindrical in shape. In FIG. 6C, thecartridge 1 also has a similar finger grip 5 to that shown by the dashedline in FIG. 6A. FIGS. 6C and 6D illustrate that the optical cell 6 insecond cartridge 1 is more elongate in shape. The elongate shape permitsgreater depth and rise of fluid for greater optical pathlength andproportionally greater detection sensitivity. In addition, it iscontemplated that the top and bottom halves 2 and 3 of each cartridge 1may comprise one or more male (female) members and one or morereciprocal and interfitting female (male) members to stack theunassembled cartridge halves one on top of the other, as well as providemating alignment in the assembled state.

The dimensions of a particular cartridge 1 may vary depending upon thenumber and/or type of assays to be performed. However, in oneembodiment, as shown schematically in FIG. 6A, for example, thecartridge 1 has a length of about 10.16 cm (4.00″), width of about 2.54cm (1.00″), and a height of about 0.476 cm (0.188″). The bore of theconduit 8 running from the fluid inlet port 4 to the optical cell 6 isabout 0.127 cm (0.050″), where the lysate is dried on a region 14 of theconduit 8 about 2.381 cm (0.938″) from the fluid inlet port 4, and achromogenic substrate is dried on a region 16 of the conduit 8 about4.65 cm (1.831″) from the fluid inlet port 4. The optical cell 6 in thisembodiment is dimensioned to accommodate about 25 μL of sample.

Specimen Collection and Preparation

The cartridge may be used to determine the level of microbialcontamination in a fluid, for example, a fluid to be administeredlocally or systemically, for example, parenterally to a mammal, or abody fluid to be tested for infection, including, for example, blood,lymph, urine, serum, plasma, ascites fluid, lung aspirants, and thelike. In addition, the cartridge may be used to determine the level ormicrobial contamination in a water supply, for example, a supply ofdrinking water. In addition, the cartridge may be used to determine thelevel of microbial contamination in a food product, pharmaceutical, ormedical device.

In general, materials used to harvest, store, or otherwise contact asample to be tested, as well as test reagents, should be free ofmicrobial contamination, for example, should be pyrogen-free. Materialsmay be rendered pyrogen-free by, for example, heating at 250° C. for 30minutes. Appropriate precautions should be taken to protectdepyrogenated materials from subsequent environmental contamination.

Representative Assays that can be Performed in the Cartridge

It is contemplated that a variety of hemocyte lysate assays may be usedin the cartridge of the invention, such as, for example, the end pointturbidometric assay, the kinetic turbidometric assay, the endpointchromogenic assay, and the single-step kinetic assay. In addition, thecartridge of the invention may be used with the multi-step kineticassay, as described herein.

1. End Point Turbidometric Assay

The end point turbidometric assay is described in Prior (1990) supra,pp. 28-34. Briefly, the end point turbidimetric assay includes the stepsof (i) solubilizing a hemocyte lysate with a sample to be analyzed, (ii)incubating the resulting mixture at a temperature of about 0° to about40° C., preferably about 25° to about 40° C., for a predetermined time,and (iii) measuring the increase in turbidity as a result ofcoagulation, if any, using a conventional coagulometer, nepherometer, orspectrophotometer.

Referring to FIG. 2A, in order to perform an endpoint turbidometricassay in a cartridge 1, a sample is moved, for example, to a firstregion 14 of the conduit 8 containing the hemocyte lysate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, turbidity, using anoptical detector. Results from multiple assays, for example, two assayscan be averaged. The optical density of the sample-lysate mixture at acertain predetermined time point may then be interpolated onto apredetermined standard curve, for example, showing turbidity values onthe Y axis versus endotoxin concentration on the X axis, to give theconcentration of the microbial contaminant in the sample.

2. Kinetic Turbidometric Assay

The kinetic turbidometric assay is described in Prior (1990) supra, pp.28-34. Briefly, the kinetic turbidimetric assay includes the steps of(i) solubilizing a hemocyte lysate with a sample to be analyzed, (ii)incubating the resulting mixture at a temperature of about 0° to about40° C., preferably about 25° to about 40° C., over a predetermined timerange, and (iii) measuring a time required for either a turbidity changecaused by coagulation to reach a pre-selected value or a ratio in changeof the turbidity, using a conventional coagulometer, nepherometer, orspectrophotometer.

Referring to FIG. 2A, in order to perform a kinetic turbidometric assayin a cartridge 1, a sample is, for example, moved to a first region 14of the conduit 8 containing the hemocyte lysate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, turbidity, by measuringthe absorbance or transmittance properties of the sample-lysate mixtureusing an optical detector. The detector may determine how long it takesfor each optical property to exhibit, for example, a 5% drop in opticaltransmittance. Results from multiple assays, for example, two assays canbe averaged. The resulting values may then be interpolated onto apredetermined standard curve, for example, showing time for apreselected change in transmittance on the Y axis versus endotoxinconcentration on the X axis, to give the concentration of thecontaminant in the sample.

3. Endpoint Chromogenic Assay

The endpoint chromogenic assay is described in Prior (1990) supra, pp.28-34, and U.S. Pat. Nos. 4,301,245 and 4,717,658. Briefly, the endpointchromogenic assay includes the steps of (i) solubilizing a hemocytelysate preparation with a sample to be analyzed, (ii) incubating theresulting mixture at a temperature of about 0° C. to about 40° C.,preferably about 25° C. to about 40° C., for a predetermined time, (iii)contacting a test device containing chromogenic substrate with theincubated sample-lysate mixture, (iv) adding a reaction inhibitor, and(v) measuring, e.g., by calorimetric change, a substance released fromthe synthetic substrate by protease activity.

Referring to FIG. 2A, in order to perform an endpoint chromogenic assayin a cartridge 1, a sample is moved, for example, to a first region 14of the conduit 8 containing the hemocyte lysate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. Following a predetermined incubation period, the sample-lysatemixture then is moved, for example, by pump action to a second region 16of the conduit 8 containing the chromogenic substrate, where it issolubilized, for example, by cycling between forward and reverse pumpaction. The sample-lysate-substrate mixture then is moved to a thirdregion containing a reaction inhibitor. Afterwards, thesample-lysate-substrate mixture then is moved to optical cell 6 formeasurement of an optical property, for example, the absorbance ortransmittance properties of the sample by an optical detector. Theoptical property of the sample-lysate-substrate mixture at a certainpredetermined time point may then be interpolated onto a predeterminedstandard curve, for example, showing absorbance, optical density, ortransmittance on the Y axis versus endotoxin concentration on the Xaxis, to give the concentration of the microbial contaminant in thesample.

4. Single-Step Kinetic Assay

A single-step kinetic assay, for example, a single step-chromogenicassay, is described in U.S. Pat. No. 5,310,657. Briefly, the kineticchromogenic assay includes the steps of (i) simultaneously solubilizinga hemocyte lysate with a sample to be analyzed and a chromogenicsubstrate, (ii) incubating the resulting mixture at a temperature ofabout 0° to about 40° C., preferably about 25° to about 40° C., over apredetermined time range, and (iii) measuring a time required for acolorimetric change to reach a pre-selected value or a ratio in changeof the calorimetric readout, using a conventional spectrophotometer.

Referring to FIG. 2A, in order to perform a kinetic chromogenic assay ina cartridge 1, a sample is moved, for example, by pump action, to afirst region 14 of the conduit 8 containing both the hemocyte lysate andchromogenic substrate, where it is solubilized, for example, by cyclingbetween forward and reverse pump action. The sample-lysate-substratemixture then is moved to optical cell 6 for measurement of an opticalproperty for example, the absorbance or transmittance properties of thesample by an optical detector. The detector may determine how long ittakes for each optical property to exhibit, for example, a 5% drop inoptical transmittance. Results from multiple assays, for example, twoassays can be averaged. The resulting values may then be interpolatedonto a predetermined standard curve, for example, showing the time for apreselected change in absorbance or transmittance (as the case may be)on the Y axis versus endotoxin concentration on the X axis, to give theconcentration of the contaminant in the sample.

Of the above methods, the endpoint chromogenic assay and the single-stepkinetic chromogenic assays currently are considered the most rapid,sensitive, and economic assays for the detection of microbialcontaminants, for example, endotoxin. However, both assays have theirlimitations. The endpoint chromogenic assay is rapid (about 15 minutes)but typically can only detect endotoxin concentrations in a range thatis limited to about one log range (for example, about 1 EU/mL to about0.1 EU/mL), with a sensitivity of about 0.1 EU/mL. Although thesingle-step kinetic chromogenic assay measures endotoxin concentrationsin a wider range of about 5 logs (for example, about 5 to about 0.05EU/mL) with a high sensitivity of about 0.05 EU/mL, this method can bequite slow to run (about 30 minutes). Furthermore, neither the endpointchromogenic assay nor the single-step kinetic chromogenic assay arereadily adaptable for in-field performance. The multi-step kinetic assayovercomes the limitations in the endpoint chromogenic assay and thekinetic chromogenic assay.

6. Multi-Step Kinetic Assay

As will be discussed in more detail, the cartridge may also be used toperform a multi-step kinetic assay. The various steps involved in themulti-step kinetic assay are shown schematically in FIG. 7. The assay isinitiated by combining the sample to be tested with a volume of ahemocyte lysate to produce a sample-lysate mixture. The mixture then isincubated for a predetermined period of time. The sample-lysate mixturethen is contacted with a substrate, for example, a chromogenicsubstrate, to produce a sample-lysate-substrate mixture. Thereafter, thetime in which a preselected change in an optical property (for example,a specific change in an absorbance value or a specific change in atransmission value) is measured. The presence and/or amount of microbialcontaminant may be then determined by interpolating the measured timeagainst a pre-calibrated standard curve, for example, a standard curveshowing the time to make a preselected change in optical property(absorbance or transmittance) on the Y axis versus endotoxinconcentration on the X axis.

The standard curve may be created, for example, by adding increasingamounts of an agent, for example, an endotoxin, glucan, or othermicrobial cell wall component, in a blank sample, for example,pyrogen-free water. The time for which a preselected change in anoptical property, for example, a preselected increase in absorbance or apreselected decrease in transmittance, is determined for eachconcentration of the microbial cell wall component. The various timemeasurements to achieve a standard change in optical property then areplotted as a function of the microbial cell wall componentconcentration. In general, the concentration of endotoxin is inverselyproportional to the time necessary to achieve the standard change inoptical property. The standard curve can then be used to assess thepresence and/or amount of microbial contaminant in the sample ofinterest. The calculation of standard curves is provided in Example 2.

As will be apparent to one skilled in the art, the relative amounts ofhemocyte lysate and substrate can be adjusted to ensure that effectiveamounts of these two components are present in thesample-lysate-substrate mixture at the end of the assay. The finalamount of hemocyte lysate protein in the assay is from about 1 μg toabout 500 μg, preferably about 50 μg. The final amount of the substrate,for example, the chromogenic substrate in the assay is from about 1 μgto about 50 μg, preferably about 6.5 μg. The determination of theconcentration and composition of the substrate, for example, thechromogenic substrate, is considered to be within the level of skill inthe art.

The final volume of the resulting sample-lysate-substrate mixture can bebased on the requirements of the optical detector used to measure thechange in optical property of the sample. The ratio of volumes betweenthe sample, lysate, and substrate can be readily established by those ofordinary skill in the art. Depending on the relative volumes of thesample, lysate, and substrate in the sample-lysate-substrate mixture,the concentration of the other components of the assay can be adjustedto maintain the final concentrations in the operable range, as describedherein.

Referring to FIG. 2A, to perform the multi-step kinetic assay in acartridge 1 of the invention, a sample is first moved, for example, bypump action, to a first region 14 containing the hemocyte lysate, whereit is mixed and incubated for a predetermined period of time. Thesample-lysate mixture then is moved, for example, by pump action, to thesecond region 16 containing the substrate, for example, the chromogenicsubstrate, where it is solubilized. The sample-lysate-substrate mixturethen is moved to optical cell 6, for a measurement of an opticalproperty. The time intervals required for mixing and incubating stepsare preprogrammed for optimal sensitivity and microbial contaminantconcentration range.

FIGS. 8A and 8B show an exemplary cartridge and hand held opticaldetector useful in the practice of the invention. FIG. 8A shows thecartridge being introduced into the detector and FIG. 8B shows thecartridge inserted into the detector with the fluid inlet ports stillexposed to the user.

FIG. 9 is a graph showing the changes in optical properties that can begenerated in a cartridge using a multi-step kinetic assay. The dashedline represents changes in transmittance of the sample over time. Thesolid line represents changes in absorbance of the sample over time.Referring to FIGS. 2A and 9, optical properties were monitored after analiquot of sample was added to the fluid inlet port 4 at a time of 0seconds. After 60 seconds, the sample was drawn to region 14, where itwas mixed with hemocyte lysate disposed on region 14 for 60 seconds(represented by vertical zig zag lines from T=60 to T=120). Theamplitude of the mixing (length of the vertical lines) is determinedsuch that the sample is repeatedly moved over the entire region 14.Thereafter, the sample-lysate mixture was incubated from 120 to 480seconds at region 14 without further mixing. After 480 seconds, thesample was drawn to a chromogenic substrate at region 16 and thesample-lysate mixture combined with the chromogenic substrate from theperiod shown from 480 to 540 seconds (represented by vertical zig zaglines from T=480 to T=540). The amplitude of the mixing (length of thevertical lines) is determined such that the sample is repeatedly movedover the entire region 16. The resulting sample-lysate-substrate mixturethen was drawn to optical cell 6 and the optical properties (absorbanceand transmittance) measured for the period from 540 second to 1440seconds.

Using the initial absorbance or transmittance readings of the mixture,the time required for the absorbance or transmittance to change by anarbitrary amount (Reaction Time) is determined. The amount of microbialcontaminant in the sample then is determined by comparing the ReactionTime for the sample against a predetermined standard curve.

A spiked sample is assayed in parallel with the unspiked sample. Themicrobial contaminant concentration in the unspiked sample and themicrobial contaminant recovered in the spiked sample can be compared todetermine the presence of interference, such as an inhibitor or anenhancer of the reaction, as previously described.

Although the multi-step assay may be performed in a cartridge of thetype discussed above, it may be employed in a variety of other formats,for example, within the well of a microtiter plate. Exemplary assaysperformed in the well of a microtiter plate are discussed in Example 3.Multiple samples of various test fluids, as well as spiked samples andthe series of control samples making up a standard curve, may be placedin the wells of the microplate. Fixed amounts of hemocyte lysate andthen substrate are added to each of the wells, preferably using anautomated system, such as a robot, and the plate processed by amicroplate reader, which can be programmed to sequentially read theabsorbance of each well in a repetitive fashion.

In addition, it is contemplated that the cartridges, glucan-specificassays, and the multi-step kinetic assays can be used to detect thepresence and/or amount of a ligand of interest in a test sample. Forexample, by adapting the assay format as appropriate, it is possible todetect the presence and/or amount of any ligand of interest, forexample, a drug, toxin, protein, metabolite, in a sample. An exemplaryligand assay performed in the well of a microtiter plate is discussed inExample 7. By way of example, a binder for a ligand of interest, forexample, an antibody or antigen binding fragment thereof, is immobilizedon the surface of a solid support, for example, in a cartridge or amicroplate. The binder is then pre-loaded or pre-bound with a complexcomprising the ligand of interest coupled or bound to lipopolysaccharideor glucan. Methods for conjugating glucan or lipopolysaccharide to aligand are well known in the art. For example, Boutonnier et al. (2001)INFECT. IMMUN. 69:3488-3493, describe methods for conjugatinglipopolysaccharide to tetanus toxoid (see also, Konadu et al. (1994)INFECT. IMMUN. 62:5048-5054; Kenne et al. (1982) CARBOHYDR. RES.100:341-349). When a sample containing the ligand of interest iscombined with the immobilized binder, the lipopolysaccharide orglucan-labeled ligand is displaced. The amount of displaced ligand canthen be quantified by the extent of the lipopolysaccharide or glucaninitiated reaction of a LAL preparation.

Using these principles, it is possible to create a cartridge fordetermining the presence and/or amount of a ligand of interest in asample. In this type of format, the binder for ligand is immobilized ona surface of the conduit downstream of the sample inlet port andupstream of the optical cell. The binder for ligand is then preloadedwith a complex comprising the ligand of interest coupled or conjugatedto, for example, lipopolysaccharide or glucan. A hemocyte lysate (forexample, a Factor G-specific lysate for detecting displaced glucan, or aFactor C-specific lysate for detecting displaced lipopolysaccharide) isimmobilized on a surface of the conduit downstream of the immobilizedbinder for ligand. When the sample of interest is applied to the sampleinlet port, the sample passes the binder for ligand. To the extent thatthe sample contains the ligand, the ligand displaces the complex fromthe immobilized binder. The amount of displaced ligand can be measuredby measuring a change in an optical property in the hemocyte lysate.This change in optical property can then be correlated with the amountof the ligand of interest in the sample.

EXAMPLES

Practice of the invention will be more fully understood from thefollowing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

Example 1 Preparation of the Chromogenic Assay Cartridge

An exemplary cartridge shown in FIG. 2 was prepared as follows.Referring to FIG. 5A, the LAL and chromogenic substrates were applied toregions 14′ and 16′, respectively, of conduit 8′ of the bottom half 2 ofthe cartridge 1 using a Hamilton Microlab 540B Dispenser (HamiltonCompany, Reno, Nev.). Briefly, 4-5.0 μL of 10 mg/mL Endosafe LAL(Charles River Endosafe, Charleston, S.C.) containing 1% mannitol(Osmitrol, Baxter, Deerfield, Ill.) and 0.1% dextran (MW 10-100K,Sigma-Aldrich, St. Louis, Mo.), was applied to regions 14′. 4-5.0 μL of(1.3 mg/mL) chromogenic substrate Ile-Glu-Ala-Arg-pNA Chromogenix S-2423(Instrumentation Laboratories, Milan, Italy) containing 1% polyvinylalcohol (PVA) (MW 7K-30K, Sigma-Aldrich, St. Louis, Mo.), was applied toregions 16′. The bottom half 2 of the cartridge 1 was dried under acontrolled temperature of 25° C.+/−2° C. and a humidity of 5%+/−5% in aLunaire Environmental Steady State & Stability Test Chamber (LunaireEnvironmental, Williamsport, Pa.) in a Puregas HF200 Heatless Dryer (MTIPuregas, Denver, Colo.) for 1 hour. Temperature and humidity wascontrolled by a Watlow Series 96 1/16 DIN Temperature Controller (WatlowElectric Manufacturing Company, St. Louis, Mo.).

Referring to FIG. 5B, 5 μl Endosafe CSE endotoxin (Charles RiverEndosafe, Charleston, S.C.) (“spike”) was applied to region 14″ of theconduit 8″ of the top half 3 of the cartridge 1. The top half 3 of thecartridge 1 was dried under a controlled temperature of 25° C.+/−2° C.and a humidity of 5%+/−5% for one hour, as described above.

Following fabrication, the two halves 2 and 3 were assembled such thatregions 14′ and 14″ were aligned one on top of the other, and the edgesof the cartridge halves 2 and 3 ultrasonically sealed using a DukaneModel 210 Ultrasonic Sealer (Dukane Corporation, St. Charles, Ill.)under the control of a Dukane Dynamic Process Controller (DukaneCorporation, St. Charles, Ill.).

The resulting cartridge 1 was labeled to identify the lot number of thecartridge, in order to later identify the standard curves used toquantify the microbial contaminant in the sample. The sealed, labeledcartridge 1 then was placed into a laminated foil pouch along with adesiccant such as silica gel or molecular sieve. The foil pouch waspurged with nitrogen gas and then sealed with a PAC Model PV-G VacuumImpulse Heat Sealer (Packaging Aids Corporation, San Rafael, Calif.).

Example 2 Cartridge-Based Assays

This example demonstrates that a cartridge of the invention can be usedto measure the amount of a microbial contaminant by an endpointchromogenic assay, kinetic chromogenic assay and a multi-stepchromogenic assay.

FIG. 8 shows how the cartridge of the invention may be used with aportable hand-held optical detector. FIG. 8A shows the cartridge in theprocess of being inserted into the optical detector. FIG. 8B shows thecartridge inserted fully into the optical detector, however, the fluidinlet ports of the cartridge are still accessible to the user. Thisconfiguration permits the user to apply one or more samples of interestto the exposed fluid inlet ports even though the optical cells of thecartridge are located in place within the optical detector.

(I) Endpoint Chromogenic Assay in a Cartridge

A cartridge was prepared essentially as described in Example 1, with theexception that no spikes were added to the conduits. Samples ofendotoxin standards of 1.0 EU/mL, 0.5 EU/mL, 0.25 EU/mL, and 0.125 EU/mLwere prepared and 25 μL of each were pipetted into one of four cartridgefluid inlet ports. The portable optical detector maintained atemperature of 37° C. for the duration of the test. The portable opticaldetector was programmed to perform a series of steps. The portableoptical detector first drew each endotoxin sample into the region of aconduit that contained dried LAL, and mixed and incubate the sample withthe LAL for 120 seconds (T=60 to T=180). The portable optical detectorthen drew the endotoxin sample-LAL mixture to the region in the conduitcontaining dried chromogenic substrate, and mixed the sample-LAL mixturewith the substrate for 5 seconds. The portable optical detector thendrew the sample-LAL-substrate mixture to the optical cell. The portableoptical detector then recorded absorbance data from each of the fourchannels. After about 10 minutes (about 780 seconds), the test was endedby reading the last absorbance (optical density) value. The absorbancevalue curves for each endotoxin sample are shown in FIG. 10A. Theabsorbance values generated at 780 seconds were recorded and plotted asa function of endotoxin concentration (FIG. 10B) to give a standardcurve. This standard curve can be used to determine the concentration ofendotoxin in a sample of interest, when the sample is treated in thesame manner as the standards.

(II) Single-Step Kinetic Chromogenic Assay in a Cartridge

A cartridge was prepared essentially as described in Example 1, with theexceptions that the chromogenic substrate was applied to the same regionas the LAL and that no spikes were added to the conduits. Samples ofendotoxin standards of 5.0 EU/mL, 0.5 EU/mL, and 0.05 EU/mL wereprepared, and 25 μL of the 5 EU/mL standard was pipetted into two fluidinlet ports of the cartridge. The portable optical detector maintained atemperature of 37° C. for the duration of the test. The portable opticaldetector was programmed to perform a series of steps. The portableoptical detector first drew each endotoxin sample into the region of aconduit that contained both dried LAL and chromogenic substrate, andmixed and incubated the sample with the LAL and substrate for 30seconds. The portable optical detector then drew thesample-LAL-substrate mixture to the optical cell. The portable opticaldetector began recording absorbance for both samples. The assay wasrepeated for the 0.5 EU/mL and 0.05 EU/mL standards.

The plots of recorded data for each endotoxin standard are shown inFIGS. 11A, 11B, and 11C. The time taken for the optical density of eachendotoxin standard-LAL-substrate mixture to reach an optical density of0.05 was recorded as the onset time for each standard. A plot of the logof the endotoxin concentration (X axis) vs. the log of the onset times(Y axis) provides a kinetic standard curve (FIG. 11D). This standardcurve can be used to determine the concentration of endotoxin in asample of interest when the sample is treated in the same manner as thestandards.

(III) Multi-Step Kinetic Assay in a Cartridge

A cartridge was prepared essentially as described in Example 1, with theexception that no spikes were added to the conduits. Samples ofendotoxin standards of 1.0 EU/mL, 0.5 EU/mL, 0.25 EU/mL, and 0.125 EU/mLwere prepared and 25 μL of each were pipetted into one of four cartridgefluid inlet ports. The portable optical detector maintained atemperature of 37° C. for the duration of the test. The portabledetector was programmed to perform a series of steps. The portableoptical detector first drew each endotoxin sample into the region of aconduit that contained dried LAL, and mixed and incubate the sample withthe LAL for 240 seconds. The portable optical detector then drew theendotoxin sample-LAL mixture to the region in the conduit containingdried chromogenic substrate and mixed the sample-LAL mixture with thesubstrate for 20 seconds. The portable optical detector then drew thesample-LAL-substrate mixture to the optical cell. The portable opticaldetector began recording absorbance data from each of the four channels.The time taken for the optical density of each endotoxinstandard-LAL-substrate mixture to reach an optical density of 0.05 wasrecorded as the onset time for each standard (see, Table 1). A plot ofthe log of the endotoxin concentrations (X axis) versus the log of theonset times (Y axis) provides a kinetic standard curve (FIG. 12). Thisstandard curve can be used to determine the concentration of endotoxinin a sample when the sample is treated in the same manner as thestandards.

TABLE 1 Endotoxin Concentration, EU/mL 5.0 0.5 0.05 0 Onset Time orReaction Time 30 166 222 300 (seconds)

Example 3 Microplate-Based Assays

This example demonstrates that a multi-well microtiter plate can befabricated so that the amount of a microbial contaminant (bacterialendotoxin in this example) can be measured by an endpoint chromogenicassay, a single-step kinetic assay and a multi-step assay.

(I) Single-Step Kinetic Assay on a Microplate

A single-step kinetic chromogenic assay was performed as follows.Briefly, 50 μL of a control microbial contaminant of interest (e.g., 5EU/mL, 0.5 EU/mL, or 0.05 EU/mL of Endosafe Control Standard Endotoxin(CSE), Charles River Endosafe, Charleston, S.C.), was added to one ormore wells of a 96 well plate. 50 μL of 5 mg/mL Endosafe LAL (CharlesRiver Endosafe, Charleston, S.C.) and 5 μL of 1.3 mg/mL ChromogenixS-2423 chromogenic substrate (Instrumentation Laboratories, Milan,Italy) were added to each well and incubated at 37° C. for 32 minutes.The optical density of the mixture in each well was monitored by aspectrophotometer, such as a Sunrise micro plate reader (Tecan, ResearchTriangle Park, N.C.). The time taken for each standard to change 0.1absorbance units was determined (“onset time”). The results aresummarized below in Table 2. A plot of the log of the endotoxinconcentrations (X axis) versus the log of the onset times (Y axis)provides a kinetic standard curve (FIG. 13). This standard curve may beused to measure the concentration of endotoxin in a sample of interest,when the sample is treated in the same manner as the standards.

TABLE 2 Time to Onset OD/ Mean Time to Concentration, Max Reaction TimeReach Onset OD Standard (RT) Calculated Standard EU/mL (seconds)(seconds) Deviation CV % Value STD1 5.0 229.4/221.4 225.4 4.0/4.01.77 >4.9853 STD2 0.5 472.2/465.0 468.6 3.59/3.59 0.77 0.5029 STD3 0.05982.5/977.1 979.8 2.68/2.68 0.27 <0.0499 CTRL1 0 >990.0/>990.0 >990.00.00/0.00 0.00 <0.0500(II) Endpoint Assay on a Microplate

An endpoint chromogenic assay using the reagents in Example 3(I) wasperformed as follows. Briefly, 50 μL of a control microbial contaminantof interest (e.g., endotoxin) at a concentration of e.g., 5 EU/mL, 0.5EU/mL, or 0.05 EU/mL, was added to one or more wells of a 96-well plate.50 μL of hemocyte lysate (5 mg/mL) was added to one or more wellscontaining the standard and incubated at 37° C. for 5 minutes. 5 μL ofchromogenic substrate (1.3 mg/mL) was added to one or more wells andincubated at 37° C. for 5 minutes. The reaction then was stopped by theaddition of 100 μL of 50% acetic aid to each well. The optical densityof the mixture in each well was measured at 405 nm by aspectrophotometer, such as a Sunrise micro plate reader (Tecan, ResearchTriangle Park, N.C.). The absorbance of each sample at 780 seconds(shown in Table 3) was recorded. The absorbance of each sample (Y axis)was plotted versus the endotoxin concentration (X axis) to provide astandard curve (shown in FIG. 14). This standard curve may be used tomeasure the concentration of endotoxin in a sample of interest, when thesample is treated in the same manner as the standards.

TABLE 3 Concentration, Optical Density Mean Optical Standard (OD)Calculated Standard EU/mL (OD) Density Deviation CV % Value STD1 1.20.7505/0.8105 0.7805 0.03/0.03 3.84 1.1876 STD2 0.6 0.4135/0.3825 0.39800.02/0.02 3.89 0.6319 STD3 0.3 0.1655/0.1615 0.1635 0.00/0.00 1.220.2912 STD4 0.15 0.0655/0.0525 0.0590 0.01/0.01 11.02 0.1393 CTRL1 0 0.0115/−0.0115 0.0000 0.01/0.01 0.00 <0.1500(III) Multi-Step Kinetic Assay on a Microplate

A multi-step kinetic chromogenic assay using the same reagents as inExample 3(I) was performed as follows. Briefly, 50 μL of a controlmicrobial contaminant of interest, for example, endotoxin, at thefollowing concentrations of 5 EU/mL, 0.5 EU/mL, or 0.05 EU/mL, was addedto one or more wells of a 96-well plate. 50 μL of hemocyte lysate (5mg/mL) was added one or more wells and incubated at 37° C. for 3minutes. 5 μL of chromogenic substrate (1.3 mg/mL) was added to one ormore wells containing the standard and incubated at 37° C. for 16.5minutes. The optical density of the mixture in each well was monitoredby a spectrophotometer, such as a Sunrise micro plate reader (Tecan,Research Triangle Park, N.C.). The time taken for each microbialcontaminant standard and/or sample to change 0.1 absorbance units wasdetermined (“onset time”). The results are summarized in Table 4. A plotof the log of the endotoxin concentrations (X axis) versus the log ofthe onset times (Y axis) provides a kinetic standard curve (FIG. 15).This standard curve may be used to measure the concentration ofendotoxin in a sample of interest, when the sample is treated in thesame manner as the standards.

TABLE 4 Time to Onset OD/ Mean Time to Concentration, Max Reaction TimeReach Onset OD Standard (RT) Calculated Standard EU/mL (seconds)(seconds) Deviation CV % Value STD1 5.0 172.1/166.4 169.3 2.84/2.841.68 >5.1505 STD2 0.5 393.8/387.7 390.7 3.03/3.03 0.78 0.4712 STD3 0.05851.7/843.3 847.5 4.17/4.17 0.49 <0.0515 CTRL1 0 >990.0/>990.0 >990.00.00/0.00 0.00 <0.0500

Example 4 Preparation and Testing of Glucan-Specific LAL

As shown in FIG. 1, crude LAL reacts with both endotoxin(lipopolysaccharide) and glucan, so that cell wall material from bothGram negative bacteria and yeast/mold cells activate the coagulationcascade. In the case of Gram negative bacteria, coagulation is mediatedthrough the Factor C cascade. In the case of yeast and mold, coagulationis mediated through the Factor G cascade. In order to determine which ofthe two contaminants is present in a sample, or what proportion of eachmight comprise a mixed contamination, LAL was produced under conditionsto render it specific for glucan, as described below.

Amebocyte blood was mixed 6:1 with 0.05% Tween 20 (Sigma, St. Louis,Mo.), 150 mM NaCl and centrifuged in a Sorval model RC-3B centrifuge at3,000 rpm for 5 minutes. The pelleted cells were washed with 0.01% Tween20, 150 mM NaCl and centrifuged at 3,000 rpm for 5 minutes. Thepelleted, washed cells were divided into three aliquots, resuspended ineither lipopolysaccharide-free water, lipopolysaccharide-free 1 M NaCl,or lipopolysaccharide-free 2 M NaCl. The cells in each pellet were lysedby sonication for 1-2 minutes. Cell debris was removed by centrifugationat 4,000 rpm for 10 minutes, and the resulting supernatant was harvestedand used directly in coagulation experiments.

FIG. 16 provides a graphical representation of kinetic coagulationreactions using various concentrations of lipopolysaccharide (FIG. 16A)or glucan (FIG. 16B) in a microtiter plate multi-step kinetic assay,such as that described in Example 3 (III) with either standard LAL (rowA of each figure) or glucan-specific LAL (rows B and C in each figure).

FIG. 16A illustrates the reactivity of the lysates withlipopolysaccharide (Charles River Endosafe) serially diluted 1:10 fromcolumn 1 to column 5. The lipopolysaccharide concentrations in columns 1through 5 are: 10 ng/ml, 1 ng/ml, 100 pg/ml, 10 pg/ml, and 0,respectively.

FIG. 16B illustrates the reactivity of the lysates with glucan (CharlesRiver Endosafe, Charleston, S.C.) serially diluted 1:10 from column 1 tocolumn 5. Glucan concentrations from column 1 through 5 are: 100 μg/ml,10 μg/ml, 1 μg/ml, 100 ng/ml, and 0, respectively.

In each figure, row A shows the reactivity with standard LAL preparedfrom cells lysed in pyrogen-free water (i.e., reactive with both glucanand lipopolysaccharide). In each figure, row B is glucan-specific LALproduced by lysing cells in 1 M NaCl. In each figure, row C isglucan-specific LAL produced by lysing cells in 2M NaCl. Each graphrepresents the change in optical density or absorbance, as shown on theY axis, over time, as shown on the X axis. For example, in FIG. 16A, thegraph shown in row A, column 1, shows the change in absorption over timewhen 10 ng/ml lipopolysaccharide is added to standard LAL.

The results demonstrate that when lipopolysaccharide is added tostandard lysate, the lipopolysaccharide activates the lysate to producean increase in absorbance (see, FIG. 16A, row A). However, the rate ofabsorbance change decreases as less lipopolysaccharide is added to eachsample. The results demonstrate, however, that there is substantially nochange in absorbance as lipopolysaccharide is added to eachglucan-specific lysate (see, FIG. 16A, rows A and B).

The results also demonstrate that when glucan is added to standardlysate, the glucan (like the lipopolysaccharide) activates the lysate toproduce an increase in absorbance (see, FIG. 16B, row A). However, therate of absorbance change decreases as less glucan is added to eachsample. In contrast to the situation when lipopolysaccharide was added,the glucan activates each glucan-specific lysate to produce an increasein absorbance over time (see, FIG. 16B, rows B and C).

These results demonstrate that it is possible to produce aglucan-specific lysate using the protocol described herein.

Example 5 Testing of Glucan-Specific LAL in a Cartridge-Based Multi-StepAssay

Glucan-specific LAL was prepared by lysing amebocytes in 2M NaCl asdescribed in Example 4 and tested in a cartridge-based multi-stepkinetic assay, such as that described in Example 2(III).

Samples containing glucan at 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml,and 0, were incubated with the glucan-specific lysate for 4 minutes. Theresulting mixture was mixed with a chromogenic substrate,acetate-Ile-Glu-Ala-Arg-pNA and the change in absorbance at 405 nm wasmeasured over time. The time required to reach an onset optical densityof 0.05 was collected (see, Table 5) and the log of the glucanconcentration was plotted versus the log of the time to reach onset O.D.(see, FIG. 17).

TABLE 5 Glucan Standard Onset Time Calculated μg/ml (μg/ml) (seconds)glucan activity 100 207 80.5  10 400 15.4  1 1300 0.80    0.1 >1800<0.36 Negative control >1800 <0.36

The data show that it is possible to produce a standard curve of glucanconcentration using a cartridge-based multi-step assay. The standardcurve can then be used to determine the concentration of glucan in asample of interest, when the sample is treated in the same manner as thestandards.

Example 6 Means of Reading an LAL Reaction Using Fluorescent Substrates

A cartridge-based multi-step kinetic LAL assay, such as that describedin Example 2(III), was performed using the fluorogenic substrate:Glu-Gly-Arg-AMC (Enzyme Systems Products, Livermore, Calif.). Thecartridge was modified to include a long-pass filter placed between thesample and the light sensor such that light having the excitationwavelength (390 nm) was blocked but yet the emissions at a wavelength of460+/−25 nm were able to pass through and be detected by the sensor.

Using the device, fluorescence data (expressed as Relative FluorescenceUnits) were collected from samples containing 10 EU (Endotoxin units), 1EU, and 0.1 EU. The changes in Relative Fluorescence Units over time arepresented in FIG. 18. The results demonstrate that the multi-stepkinetic assays can measure the amount of endotoxin in sample using afluorescent substrate.

Example 7 Measurement of Lipopolysaccharide-Labeled Ligand Using anImmobilized Antibody

The microtiter plate multi-step kinetic LAL assay, such as thatdescribed in Example 3(III) can also be used to measure theconcentration of a ligand. Briefly, an antibody that binds a ligand ofinterest is immobilized onto the surface of a well of a microtiterplate. Then, the ligand binding sites of the antibody are preloaded witha complex comprising the ligand coupled to lipopolysaccharide. When asample containing the ligand is exposed to the immobilized antibody, thelipopolysaccharide-labeled ligand is displaced and quantified byreaction with LAL. In this example, the ligand detected was fluorescein.An anti-fluorescein antibody was immobilized onto the surface of a welland was pre-loaded with fluorescein-labeled lipopolysaccharide. Whensamples containing fluorescein-labeled antibody were exposed to theimmobilized antibody, the fluorescein-lipopolysaccharide conjugate wasreleased from the immobilized antibody and measured by reactivity withLAL.

Briefly, rabbit anti-fluorescein antibody (Virostat, Portland Me.) wasdiluted 1/4000 in CAPS buffer, pH 10.2 (Sigma, St. Louis, Mo.). 25 μl ofthe diluted antibody was added to the wells of a high binding plate(Corning 25801) and incubated for 1 hour at 37° C. The plate was washed4×100 μl per well with TTBS (0.1% Tween, 100 mM Tris buffered saline)using a multipipettor. The wells were blocked by adding 150 μl per wellof gelatin diluent and stored at 4° C. overnight. The wells then werewashed with 3×100 μl per well with TTBS. Lipopolysaccharide(LPS)-fluorescein (FITC) conjugate (List Biological Laboratory,Campbell, Calif.) was diluted from 1 μg/ml to 10 pg/ml using 10-folddilutions in 0.1 M Tris. 0.1 M Tris was used as a control. 75 μl ofdiluted LPS/FITC conjugate was added per well and incubated for 30minutes at 37° C. The wells then were washed with 3×100 μl per well with0.1 M Tris.

Fluorescein-labeled goat anti-chicken antibody (Southern Biotech.Associates Inc 6100-02, Birmingham, Ala.) was diluted 1/50, 1/150, and1/450 in 0.1 M Tris. 0.1 M Tris was used as a control. 75 μl offluorescein-labeled goat anti-chicken antibody was added per well andincubated for 30 minutes at 37° C. 50 μl was removed from each well andtransferred to a clean 96-well plate (Falcon, 353072, Becton Dickenson,Franklin Lakes, N.J.). 50 μl Endochrome K (Charles River Endosafe),which is a 1:1 mixture of LAL substrate and LAL lysate, was added toeach well. The plate was read at time intervals (e.g., kinetically) at405 nm at 37° C. for 60-90 minutes (Min OD: 0, Max OD: 0.8, Onset OD:0.1).

To find the proper concentration of LPS/FITC to be pre-bound to theimmobilized antibody, increasing concentrations were applied to theplate from 10 pg/mL to 1 μg/mL. Then dilutions of thefluorescein-labeled antibody ligand were exposed to the immobilizedantibody and the displaced LPS/FITC measured as EU equivalents. As shownin FIG. 19, at the 1 μg/mL level of LPS/FITC, an EU proportional to theligand was achieved (far left). Accordingly, by using this type offormat, it is possible to detect the presence and/or measure the amountof a ligand of interest in a test sample.

Equivalents

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

Incorporation by Reference

All publications and patent documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if the entire contents of each individual publication orpatent document was incorporated herein.

1. A method of determining the presence of a microbial infection orcontamination in a sample, the method comprising the sequential stepsof: (a) contacting a sample with an amebocyte lysate comprising anactivatable enzyme to produce a sample-lysate mixture, whereupon theenzyme becomes activated if a microbial contaminant that activates theenzyme is present in the sample; (b) after step (a), contacting thesample-lysate mixture with a substrate for the enzyme to produce asample-lysate-substrate mixture, such that, if the mixture containsactivated enzyme, the activated enzyme produces a change in thesubstrate; (c) determining the time in which a preselected change occursin an optical property of the sample-lysate-substrate mixture, whereinthe change in optical property results from a change in the substrate;and (d) comparing the time determined in step (c) against apredetermined standard curve to determine whether the microbialinfection or contamination is present in the sample.
 2. The method ofclaim 1, wherein the microbial contaminant is selected from the groupconsisting of a lipopolysaccharide, a bacterial endotoxin, and a glucan.3. The method of claim 2, wherein the microbial contaminant is alipopolysaccharide.
 4. The method of claim 2, wherein the microbialcontaminant is a bacterial endotoxin.
 5. The method of claim 2, whereinthe microbial contaminant is a glucan.
 6. The method of claim 1, whereinthe change in optical property is an increase in absorbance of light ofa preselected wavelength.
 7. The method of claim 1, wherein the changein optical property is a decrease in transmission of light of apreselected wavelength.
 8. The method of claim 1, wherein the amebocytelysate is a Limulus amebocyte lysate.
 9. The method of claim 1, whereinthe amebocyte lysate is an endotoxin-specific amebocyte lysate.
 10. Themethod of claim 1, wherein the amebocyte lysate is an glucan-specificamebocyte lysate.
 11. The method of claim 1, wherein the activatableenzyme is pro-clotting enzyme.
 12. The method of claim 1, wherein theactivatable enzyme is clotting enzyme.
 13. The method in claim 1,wherein the substrate is a chromogenic substrate.
 14. The method ofclaim 13, wherein the chromogenic substrate comprises apara-nitroaniline chromophore.
 15. The method of claim 13, wherein thechromogenic substrate comprises Ile-Glu-Ala-Arg-pNA, wherein pNA is apara-nitroaniline group.
 16. The method of claim 1, comprising theadditional step of measuring the amount of the microbial contaminant inthe sample.
 17. The method of claim 1, wherein the presence of themicrobial contaminant is indicative of a microbial infection ormicrobial contamination.
 18. The method of claim 16, wherein the amountof the microbial contaminant is indicative of a microbial infection ormicrobial contamination.
 19. The method of claim 17 or 18, wherein themicrobial infection is a bacterial, yeast, mold, or fungal infection.20. The method of claim 1, wherein steps (a) and (b) provide apredetermined assay sensitivity and duration.
 21. The method of claim 1,wherein steps (a) and (b) are performed in a well defined by a solidsupport.